The present invention relates generally to a method for determining the effectiveness of antimicrobial agents against bacteria. More particularly, the invention relates to a method for rapidly determining the effectiveness of antimicrobial agents against bacteria in physiological fluids.
A rapid and routine procedure for the determination of the effectiveness of known antimicrobial agents against various bacteria is very frequently of vital importance, particularly for the measurement of the effectiveness of antimicrobial agents against bacteria in physiological fluids such as urine. Present techniques for the determination of microbial susceptibility generally require at least overnight incubation of an infecting organism after it is first isolated and cultured. The prior art techniques, therefore, require a waiting period of two days because of the double culture requirement to test the effectiveness of antimicrobial agents after the fluid specimen is received for testing.
The most commonly used conventional techniques for the determination of microbial susceptibility to antimicrobial agents include agar diffusion (the Kirby-Bauer technique), broth dilution (Mic-Broth Dilution) and agar dilution. A long standing need continues to exist in practice for a rapid technique for determining microbial sensitivity to antimicrobial agents. A testing procedure is desirable therefore, which will allow the immediate treatment of a patient with a selective antibiotic or antibiotics which excludes the inclusion of inappropriate, unnecessary, and often toxic agents in the therapeutic regimen of an infected person.
The agar diffusion (Kirby-Bauer technique) method involves an overnight culture of a urine sample. The colonies of interest are separated out and an inoculum is obtained by dilution in trypticase soy broth (TSB), to such a concentration that a dense growth is observed. The culture turbidity is adjusted to a concentration which conforms to a turbidity standard. The standard is formed by mixing 0.5 ml of 0.048 M barium chloride (1.175% w/v Ba Cl.sub.2 --2H.sub.2 O) with 99.5 ml of 0.36 (NH.sub.4).sub.2 SO.sub.4 (1% w/v). A sterile cotton swab is soaked in the diluted culture, and an agar plate is then streaked in four directions to obtain an even and thorough distribution of the organisms on the plate. The treated plates are dried for 15 minutes at 37.degree. C. Thereafter, antibiotic discs are applied to the surface of the agar and pressed into place. The plates are then allowed to stand at room temperature for 30 minutes and incubated at 37.degree. C. for 18 to 20 hours. A clearly defined zone will appear around an antibiotic disc where no bacterial growth has occurred. The zone diameter will vary with the relative resistance or susceptibility of the bacterial strain under test to the particular antibiotic used. The test must be replated for each original bacterial colony of interest. The chief disadvantage of this type of test is the time consumed to determine susceptibility.
The broth dilution (MIC-Broth Dilution) method of determing microbial sensitivity to antimicrobial agents involves visual detection of the least amount of antimicrobial agent required to cause complete inhibition of bacterial growth in a culture medium. In this technique, two-fold dilutions of antibiotics are made in a suitable culture medium. A control tube containing the culture medium, without an antibiotic, is also included for each organism tested. The organism is then allowed to grow to a logarithmic or early stationary phase of growth in the medium, and then diluted to a solution containing 10.sup.4 to 10.sup.5 viable units per milliliter. A quantity of the cultured medium is then placed in each tube of a series containing varying amounts of an antibiotic, and the tubes are allowed to incubate at 37.degree. C. for 16 to 20 hours. Thereafter, the end point of the test is determined visually, as described above. A disadvantage of this technique, in addition to the relatively long time required to complete the test is that care must be taken to recognize that slight amounts of turbidity present may be caused by the inoculum itself and not by the growth of the organism.
The agar dilution method of determining microbial sensitivity to antimicrobial agents involves the same process as the broth dilution except that the process is modified to include the introduction of nutrient agar to the antibiotic dilutions, which become solid, so that a spot application of the diluted broth culture will result in the growth or no growth of the organisms on the agar surface.